SUMMARY

The heat shock proteins (Hsps) play a positive role in lifespan
determination, and histone acetylation has been shown to be involved in
transcription of hsp genes in Drosophila. To further
determine if hsp22 and hsp70 expression is correlated with
lifespan, and if histone acetylation participates in this process, RNA levels
for hsp22 and hsp70 were analyzed throughout the lifespan in
the long-lived and short-lived iso-female lines. The results showed that
hsp22 and hsp70 RNA levels were higher in long-lived line
than in short-lived line and that the long-lived flies responded more rapidly
to heat but were more tolerant to high temperature. Moreover, we investigated
the influences of histone acetylation modification on longevity and on
hsp gene expression by using histone deacetylase (HDAC) inhibitors
TSA and BuA. The results demonstrated that both inhibitors were able to extend
the lifespan and promote hsp22 and hsp70 expression.
However, the optimal concentrations of these inhibitors, and probably the
mechanisms of their actions, vary with the genetic background. In addition, we
showed that HDAC inhibitors caused the hyperacetylation of core histone H3,
implicating the involvement of chromatin modulation in hsp gene
transcription. These data suggested a close correlation among histone
acetylation, hsp gene expression and longevity in D.
melanogaster.

Introduction

The lifespan of an organism is influenced by both genetic and environmental
factors. To date, our understanding of the mechanisms of aging is limited
because of the biological complexities of the process. There has been evidence
to support the hypothesis that aging is associated with the accumulation of
abnormal and/or malfolded proteins and oxidatively damaged proteins in many
organisms, including nematodes, flies and mammals
(Gershon and Gershon, 1970;
Stadtman, 1992). The heat
shock proteins (Hsps) are induced in response to protein damage caused by heat
and other stresses (Parsell and Lindquist,
1993). Induction of hsp genes during Drosophila
aging might have a beneficial effect on the lifespan of flies
(Wheeler et al., 1995;
King and Tower, 1999). Mild
heat stress of Drosophila transgenic for extra copies of the
hsp70 gene produced increased expression of the gene and extended the
lifespan (Tatar et al., 1997;
Tatar, 1999). Heat-induced
expression of hsp70 may reduce age-related mortality rates
(Minois et al., 2001) and
hsp22 and hsp23 genes were upregulated in selected lines for
increased longevity (Kurapati et al.,
2000), implicating the role of Hsp proteins in longevity
determination. Molecular chaperones must be playing important roles in
maintaining cellular functions during aging, through promoting protein
renaturation and preventing proteins from aggregating and denaturing
(Hartl, 1996;
Marin et al., 1993).

Expression of hsp genes represents a special model of gene
regulation involving basal and inducible expression. The
acetylation/deacetylation modifications of N-terminal tails of core histones
play critical roles in activation/repression of many eukaryotic genes,
including hsp genes. In vitro, histone acetylation has been
shown to be involved in hsp gene activation
(Nightingale et al., 1998;
Reid et al., 2000). Li et al.
(1998) demonstrated that in
Xenopus oocytes, p300 participated in the inducible transcription of
the hsp70 gene both as a coactivator and an acetyltransferase. During
Xenopus development, the presence of histone deacetylase (HDAC)
inhibitors enhanced the heat shock-induced accumulation of both hsp70
and hsp30 mRNA in post-midblastula transition (MBT) staged embryos
and resulted in the expression of hsp30 immediately after MBT rather
than at the late neurula/early tailbud stage under normal conditions
(Ovakim and Heikkila, 2003).
In a previous study, we showed that HDAC inhibitors trichostatin A (TSA) and
sodium butyrate (BuA) were able to affect the chromatin structure at the site
where the hsp70 gene is located along the polytene chromosome and
significantly enhanced both the basal and the inducible expression of
hsp70 gene in Drosophila
(Chen et al., 2002).

Histone acetylation has also been shown to be involved in lifespan
determination. In Saccharomyces cerevisiae, sir2 and rpd3,
which encode two important histone deacetylases, were found to be involved in
the regulation of lifespan, and deletion of sir2 shortened the
lifespan (Kaeberlein et al.,
1999), while the rpd3 knockout increased lifespan
(Kim et al., 1999). Additional
copies of sir2 in Caenorhabditis elegans also increased the
lifespan of the worm (Tissenbaum and
Guarente, 2001). The lifespan extension was achieved by feeding
the HDAC inhibitor phenylbutyrate (Kang et
al., 2002), or by a hypomorphic mutation of rpd3
(Rogina et al., 2002) in
Drosophila. These results indicate that histone acetylation is
associated with lifespan. But the molecular mechanisms of HDAC-dependent
changes in aging and lifespan remain unclear.

The aim of this study was to investigate the roles of histone
hyperacetylation in expression of hsp genes during aging and
longevity determination in Drosophila. Our results show that the
increase in acetylation level of histone H3 enhances the basal and inducible
expression of hsp22 and hsp70 during aging, and extends both
mean and maximum lifespan, to variable extents, in different lines of
Drosophila. This strongly implicates a correlation among histone
acetylation, hsp gene expression and longevity determination.

Materials and methods

Drosophila stocks and culture

Isofemale lines screening and lifespan determination

Eggs from a single female fly were collected and used for the screening of
iso-female lines, and ten iso-female lines were established and the lifespan
was measured. The long-lived and short-lived lines were then screened from
these lines. To obtain flies of defined age, newly eclosed flies were
collected and maintained at 25°C at a density of 50 per vial and were
transferred to fresh vials every 3 days toprevent growth of bacteria or mold,
and 200 flies for each line were cultured. The number of dead flies was
counted every day.

HDAC inhibitor treatment and heat shock induction

The HDAC inhibitors trichostain A (TSA; Sigma, MI, USA) and sodium butyrate
(BuA; Sigma) were used at final concentrations of 10 μmol l-1
and 10 mmol l-1, respectively. The third instars larvae were
divided into equal aliquots for different treatments. Part of the larvae were
cultured for 5 h in physiological brine (130 mmol l-1 NaCl, 4.7
mmol l-1 KCl and 1.9 mmol l-1 CaCl2) and then
allowed to develop directly into flies. For the repeated mild heat shock
(RMHS), the flies were induced at 32°C for 1 h every 3 days. Some of the
larvae were heat shocked (HS) at 37°C for 30 min before they were allowed
to develop into flies. For HDAC inhibitor treatment, the larvae were cultured
in physiological brine with TSA or BuA and then allowed to develop into flies
either without additional inhibitor feeding (o.TSA and o.BuA) or were
continuously cultured on medium containing inhibitors (c.TSA and c.BuA). The
remaining flies were treated with both HDAC inhibitors and RMHS (TSA-RMHS and
BuA-RMHS).

Real-time quantitative PCR analysis of hsp mRNAs

The flies were cultured and treated as described above until 6 and 30 days
old, or induced at 37°C for 2, 5, 10, 20 and 40 min. Total RNA was
extracted from flies using the RNA extraction kit supplied by Promega. The
reverse transcription (RT) reaction was performed by using a RT system
(Promega) following the manufacturer's protocol. Quantification of mRNA was
performed using an ABI PRISM® 7700 sequence Detection System (PE Applied
Biosystems, Weiterstadt, USA) and SYBR® Green (PE Applied Biosystems) as a
double-stranded DNA-specific fluorescent dye. Rp49 (ribosomal protein
49) was used as a housekeeping gene for standardizing hsp mRNA
expression.

The Ct (threshold cycle) was defined as the number of cycles required for
the fluorescence signal to exceed the detection threshold. Data were analyzed
by using the 2-ΔΔCt method, which is a convenient way
to analyze the relative changes in gene expression
(Livak and Schmittgen, 2001).
Quantitative results are given as means ±
s.e.m.

Results

Screening of the long- and short-lived lines

Longevity is dependent on both genetic and environmental factors. Under the
same environmental conditions, diversity of longevity occurs among the
different individuals. We established 10 iso-female lines of Drosophila
melanogaster, and the number of dead flies was counted every day. From
these lines, we selected the long-lived line iso2 and the short-lived line
iso4. The maximum and mean lifespan of the long-lived iso2 line were 73 days
and 45.53 days, respectively (Fig.
1). In comparison, the maximum and mean lifespan of the
short-lived iso4 line were 62 days and 38.47 days, respectively
(Fig. 1).

Basal expression of hsp genes during aging in long- and
short-lived lines

A real-time quantitative PCR procedure was used to analyze the RNA
expression of hsp genes in long- and short-lived lines during aging.
It is clear from Fig. 2 that
the hsp22 (Fig. 2A)
and hsp70 (Fig. 2B)
basal expression declined with aging and this decline was more distinct in the
long-lived line than in the short-lived line
(Fig. 2). On the other hand,
hsp22 and hsp70 mRNA levels were higher in the long-lived
line relative to the short-lived line in young flies
(Fig. 2), but became less
distinct in old flies (Fig. 2).
No prominent differences were seen in the expression of other hsp
genes, such as hsp26, during aging and between long- and short-lived
lines (data not shown).

Difference in basal expression of hsp22 (A) and hsp70 (B)
genes during aging between long- and short-lived lines. Total RNA was isolated
from flies, which were cultured and treated as described in the Materials and
methods until 6 days and 30 days. Quantitative real-time PCR was performed to
determine the mRNA expression levels of hsp genes (N=3).
Values are means ± s.e.m.

Differences in response to heat induction and in the level of
expression of hsp between long- and short-lived lines

The hsp RNA expression after 37°C heat induction for different
periods of time was also examined by real-time quantitative PCR. As shown in
Fig. 3, differences in the
response to heat induction and in the level of expression of hsp22
(Fig. 3A) and hsp70
(Fig. 3B) gene products in fly
strains with different lifespan were observed. The long-lived line was more
sensitive to heat shock than the short-lived line. Heat shock for 2 min caused
rapid increase in the expression of hsp22 and hsp70 in
long-lived line (Fig. 3, iso2),
but in the short-lived line, a detectable change in hsp22 and
hsp70 expression did not occur until 5 min of heat shock
(Fig. 3, iso4). Also,
hsp22 expression increased by approximate 130-fold in short-lived
flies at 40 min of induction, while only an approximate 80-fold increase was
detected in long-lived flies at the same time
(Fig. 3A). It can also be seen
from Fig. 3B that the intensity
of hsp70 gene did not change significantly upon heat treatment.

Difference in response to heat induction and in the level of expression of
hsp22 (A) and hsp70 (B) genes between long- and short-lived
lines. The flies were heat induced at 37°C for 2, 5, 10, 20 and 40 min
before RNA was extracted for reverse transcription. Quantitative PCR was
performed to determine the inducible mRNA expression levels of hsp
genes (N=3). Values are means ±
s.e.m. **P<0.01
versus control; †130-fold increase compared to the
control; ‡80-fold increase compared to the control.

Difference in stress resistance between long- and short-lived
lines

The experimental results described above revealed a correlation between
hsp gene expression and longevity in flies. We then investigated the
tolerance of flies to persistent high temperature, and we found that the
long-lived flies had a greater resistance to high temperature stress. Within
30 min of the treatment at 37°C rapid death occurred in the short-lived
line, whereas in long-lived flies, the rapid death occurred after 60 min of
the heat treatment (Fig.
4).

The death curves of adult flies maintained under high temperature. One
hundred flies 6 days after eclosion were kept at 37°C and the number of
dead flies was counted at each of the indicated time points for long-lived
(iso2) and short-lived (iso4) lines. Values shown are the means ±
s.e.m. of four parallel experiments.

Influence of heat shock and HDAC inhibitors on the lifespan of
flies

To test the action of HDAC inhibitors (TSA and BuA) and heat shock in flies
with different longevity, we cultured and treated flies as described in
Materials and methods, and the lifespan was measured. The repeated mild heat
shock (RMHS) resulted in an extension of both the mean and maximum lifespan of
the flies (Fig. 5A,E), but the
degrees of extension were different between the long- and short-lived lines.
The maximum lifespan increased only slightly, by 5.6%, in long-lived flies,
and moderately, by 11.5%, in short-lived flies; the mean lifespan increased
moderately, by 10.3%, in long-lived flies and considerably, by 25.8%, in
short-lived flies compared with the control. It can be seen from
Fig. 5B,F that the HDAC
inhibitor TSA strikingly influenced the lifespan in both long- and short-lived
lines, but with variable degrees. For the long-lived flies, one-off TSA
treatment (o.TSA) had no obvious effect on both mean and maximum lifespan,
while continuous TSA treatment (c.TSA) only increased mean lifespan by 15.6%
(Fig. 5B). For short-lived
flies, o.TSA resulted in an extension of mean lifespan by 5%, and c.TSA
significantly increased mean lifespan by 24.4% and maximum lifespan by 16.4%
(Fig. 5F). Data in
Fig. 5C,G demonstrated that BuA
treatment only affected the lifespan of the short-lived line, as one-off BuA
treatment (o.BuA) increased the mean lifespan by 25.8% and the maximum
lifespan by 11.5%. When BuA was added continuously throughout their lifetime
(c.BuA), the life extension effect became less obvious
(Fig. 5G). When the flies were
fed with HDAC inhibitor and RMHS was applied every 3 days, we found no
significant differences among RMHS, TSA-RMHS and BuA-RMHS treatments, and a
similar extension to that of the controls was seen
(Fig. 5D,H).

In order to test if changes in histone acetylation are involved in HDAC
inhibitor-mediated lifespan extension and hsp gene induction, we
examined the acetylation levels of core histones (H3 and H4) after HDAC
inhibitor treatment and heat shock using antibodies against acetylated H3 and
H4. As shown in Fig. 6,
acetylated lysine of histone H3 were detected. The photodensitometric analysis
of the bands revealed that both TSA and BuA caused an increase in the
acetylation level of H3 in long-lived (Fig.
6A) and short-lived (Fig.
6B) lines. More specifically, o.TSA and o.BuA treatments
moderately increased the acetyl-H3 level (P<0.05)
(Fig. 6C, o.BuA and o.TSA).
c.TSA treatments brought about a significant increase (P<0.01)
(Fig. 6C, c.TSA), whereas c.BuA
treatment moderately increased the acetyl-H3 level in long-lived flies
(P<0.05), and significantly increased it in the short-lived line
(P<0.01) (Fig. 6C,
c.BuA). Moreover, RMHS also increased acetyl-H3 (P<0.01)
(Fig. 6C, RMHS). When a
combined treatment of both HDAC inhibitors and heat shock was applied, there
was an additive effect (P<0.01)
(Fig. 6C, TSARMHS and BuARMHS).
However, no changes were detected in the acetylated lysine of histone H4 under
the same treatments (data not shown).

Hyperacetylation of histone H3 following HDAC inhibitor treatment and heat
shock induction. (A,B) Western blots of the acetylated histone H3 (AcH3) in
long-lived and in short-lived flies, respectively. The results of
photodensitometric analysis of A and B are shown in C. The upper bands are the
internal reference actin, and the lower bands are acetylated histone H3.
Values shown are the mean ± s.d. of three
independent experiments. *Significant (P<0.05);
**highly significant (P<0.01). Abbreviations as in
Fig. 5.

Next, we wanted to know whether the hyperacetylation of histone H3
influences hsp expression during aging. The hsp mRNA levels
were assayed by quantitative real-time PCR, and it was found that after c.TSA
and o.BuA treatments, hsp22 and hsp70 basal expression
increased moderately in young flies (6-day old) of the long-lived line
(P<0.05) (Fig.
7A,C, iso2), while it increased considerably in young flies (6-day
old) of the short-lived line (P<0.01)
(Fig. 7A,C, iso4). With the
aging of the flies, the degree of increase in these gene products declined
gradually. In 30-day old short-lived flies, only the basal transcription of
hsp22 increased moderately compared with control (P<0.05)
(Fig. 7B, iso4). For the
inducible expression of hsp genes, only TSA treatment gave rise to
the noticeable effect (Fig.
7E-H). In both long- and short-lived young flies, TSA-RMHS
treatment caused a higher expression of hsp22 and hsp70 than
RMHS (Fig. 7E,G). Similarly,
the increase in inducible hsp expression declined gradually during
aging (Fig. 7E-H). Until 30
days after eclosion, TSA-RMHS treatment only increased hsp22
expression in the short-lived line (Fig.
7F, iso4), while no increase in hsp70 expression was
detected (Fig. 7H). In
addition, to determine whether changes in mRNA levels actually reflect changes
in the relative levels of proteins, we assayed Hsp22 protein content in young
flies after HDAC inhibitor treatment and heat shock. Total protein was
isolated from 6-day-old flies and Hsp22 protein was detected by western blot
(Fig. 8). Consistent with
changes in the hsp22 mRNA level, Hsp22 protein expression was
up-regulated by the HDAC inhibitors (Fig.
8, c.TSA and o.BuA). Similarly, continuous TSA treatment (c.TSA)
and one-off BuA treatment (o.BuA) strikingly increased Hsp22 protein basal
expression and only TSA treatment produced a rise in inducible expression of
Hsp22 (Fig. 8, TSA-RMHS).
Additionally, it is noticeable that the HDAC inhibitor-induced up-regulation
of Hsp expression was more prominent in the short-lived line than in the
long-lived line (Fig. 8, iso2
and iso4).

Influence of HDAC inhibitor-induced histone H3 hyperacetylation on Hsp22
protein level. Western blot analysis of Hsp22 in long-lived (iso2) and
short-lived flies (iso4). The upper bands are actin used as the internal
reference and the lower bands are Hsp22 protein. Experiments were performed in
triplicate. The abbreviations as in Fig.
5.

In conclusion, this study clarifies the correlation between the elevated
expression of hsp genes and the longevity in Drosophila
melanogaster. The results revealed the higher hsp basal
expression level, higher thermotolerance and higher response to heat shock,
but lower hsp22 induction under continuous high temperature in
long-lived than in short-lived flies. Drosophila lifespan could be
extended by feeding the flies with HDAC inhibitors TSA and BuA, and/or
repeated mild heat shock. The range of lifespan extension differed between
long- and short-lived lines. HDAC inhibitor treatment promoted hsp
basal expression, but with the time of aging, the extent of this increase
declined gradually. Chromatin modulation may be involved in HDAC
inhibitor-mediated hsp gene activation, since hyperacetylation was
detected in core histone H3 upon TSA and BuA treatments. Thus, a close
correlation among histone acetylation, hsp gene regulation and aging
in D. melanogaster can be established.

Discussion

Correlation between hsp gene expression and
longevity

It has been shown that aging is associated with the accumulation of
inactive enzymes, partially denatured and/or damaged proteins
(Gershon and Gershon, 1970;
Stadtman, 1992). Heat shock
proteins are thought to reduce protein denaturation and aggregation,
facilitate re-folding of partly denatured proteins, direct the entry of
damaged proteins into proteolytic pathways and protect the organisms from
additional stress (Wheeler et al.,
1995; Parsell and Lindquist,
1993). Results presented in this report indicated that
hsp22 and hsp70 genes were upregulated and more promptly
induced in the long-lived iso-female line (Figs
2 and
3). Also, the thermotolerance
was higher in the long-lived line (Fig.
4). Lin et al.
(1998) demonstrated that the
long-lived D. melanogaster mutant displayed resistance to starvation,
high temperature and oxidative stress. A similar increased resistance to heat
was observed in long-lived C. elegans lines
(Lithgow et al., 1995). It is
possible that higher expression of hsp genes may help maintaining
cellular functions during aging and prolong the lifespan of the fly.
Meanwhile, in long-lived flies, the higher response to heat shock perhaps
results in the higher thermotolerance. On the other hand, both the basal
expression and induction speed of hsp genes in short-lived flies were
low. When the flies were kept in harsh conditions, such as high temperature,
more Hsp22 was induced to repair the damage
(Fig. 3A). This process
presumably consumes more energy and therefore results in quicker death.

Previously, two patterns of hsp expression during aging were
reported. With Oregon-R wild-type and W118 flies cultured
at low density in culture bottles, hsp22 RNA levels were low or
undetectable in young flies, but hsp22, hsp23, hsp70 were upregulated
with age (King and Tower,
1999). In contrast, when the same Oregon-R flies were cultured at
high density using population cage protocol, the starting level of
hsp22 RNA in young flies was high, and the increase in hsp22
RNA during aging was minor or undetectable
(Kurapati et al., 2000). In
the experiments of this study, Canton-S wild-type flies were cultured using
the high-density population cage protocol. In contrast to the previous
observations, we found that hsp22 and hsp70 mRNA levels were
relatively higher in young flies, and declined with age
(Fig. 2). We presume that this
may be partially due to the different strain used. Also, it is important to
note that this difference was observed under conditions that were more
stressful than the optimum.

Moreover, data in this study revealed that the response of hsp
genes to heat induction varied in flies with different longevity. In the
long-lived flies, hsp genes were more sensitive to heat than in the
short-lived line. In addition, different hsp genes exhibited a
variable response to heat, i.e. hsp22 was more responsive to heat
than hsp70. This suggests that a more positive correlation may exit
between hsp22 and longevity, while hsp70 may affect
longevity to a lesser extent. In contrast, the expression of hsp26
did not differ significantly in expression between long- and short-lived flies
(data not shown), implying that it may not be closely related to longevity.
These results implicate the existence of a mechanism for preferential
induction of hsp genes during aging.

The mechanisms of hsp gene induction by heat stress in
Drosophila have been studied in some detail
(Lis and Wu, 1993). Binding
sites for the GAGA transcription factor and other cis-acting
sequences at hsp promoters are required for generation of an
accessible promoter structure in unstressed cells, which involves a
transcriptionally engaged RNA polymerase paused ∼25 bp downstream of the
start site for transcription (Gilmour and
Lis, 1986; Lee et al.,
1992; Rougvie and Lis,
1988). Upon heat shock, the heat shock factor (HSF) is converted
into an active trimer from inactive monomers, and binds to the heat shock
element (HSE), facilitating the transcription of hsp genes. However,
the role of histone acetylation in transcription regulation of hsp
genes is still obscure. In this study, we showed that the acetylation level of
H3 was increased upon treatment with the HDAC inhibitors, TSA and BuA
(Fig. 6). We also demonstrated
that hsp genes were upregulated after HDAC inhibitor treatment
(Fig. 7). Interestingly, we
noticed that heat shock also increased the acetylation level of H3
(Fig. 6), further confirming
the involvement of histone acetylation in hsp gene transcription in
D. melanogaster. TSA and BuA are the two widely used HDAC inhibitors,
but they may function through different mechanisms
(Gibson, 2000;
Finnin et al., 1999). TSA was
originally identified as a fungal antibiotic with differentiation inducing
properties, and it can strongly increase the acetylation level of histone H3
(Zhong et al., 2003). BuA is a
simple chemical that can raise the acetylation level of H3 and H4 by
suppressing HDAC activity via a noncompetitive mechanism
(Gibson, 2000). TSA and BuA
affected hsp gene expression differently in this study. TSA was able
to induce the expression of hsp22 and hsp70 genes above
their basal level, and it worked cooperatively with heat shock to raise the
inducible transcription intensity of hsp22 and hsp70 genes
(Fig. 7). BuA only promoted the
basal expression of hsp genes
(Fig. 7A,C). In addition,
continuous TSA treatment (c.TSA) and one-off BuA treatment (o.BuA) increased
hsp22 and hsp70 expression more in short-lived flies, and
this result was consistent with the influences of these HDAC inhibitors on the
longevity of the flies (Fig.
5F,G). Moreover, as aging proceeded, HDAC inhibitor treatments
became less effective in promoting hsp gene expression. This may
presumably be due to the increasingly inactive response and repair mechanisms
in aged flies.

Histone acetylation/deacetylation modifications and the
longevity

A balance of activation and repression of various genes regulates an
optimal physiological and cellular environment for longevity. The
acetylation/deacetylation modifications of histones play critical roles in
activation/repression of many genes, and hence probably regulate the lifespan.
In yeast and worms, the HDAC Sir2 and Rpd3 have been shown to be closely
related to lifespan (Kaeberlein et al.,
1999; Kim et al.,
1999; Tissenbaum and Guarente,
2001), and feeding of the HDAC inhibitor phenylbutyrate
(Kang et al., 2002) or a
hypomorphic mutation of rpd3
(Rogina et al., 2002) in the
Drosophila extended the lifespan. However, mutation in Drosophila
sir2 (dsir2) gene did not shorten lifespan, as predicted from
yeast and worms (Newman et al.,
2002). It appears that the influence of histone acetylation on
longevity is a complex process and may involve different pathways and
mechanisms. We treated flies with a one-off and continuous feeding of HDAC
inhibitors, heat shock, as well as combinations of both HDAC inhibitors and
heat shock, in an attempt to elucidate the effects of HDAC inhibitor-mediated
histone acetylation on longevity. The results showed that TSA strikingly
extended the lifespan in different ways, as o.TSA treatment only increased the
mean lifespan in the short-lived line, and c.TSA treatment extended the mean
lifespan by 15.6% in the long-lived line; while in the short-lived line, it
extended both the mean and maximum lifespan by 24.4% and 16.4%, respectively
(Fig. 5B,F). Thus, a prolonged
TSA action in vivo is probably necessary for longevity determination.
However, BuA had less effect on lifespan. Only in the short-lived line did
o.BuA treatment extend both the mean and maximum lifespan. When BuA was
applied throughout the lifetime of the flies, its life-extension effect became
insignificant (Fig. 5G). We
presume that prolonged BuA feeding may be toxic, which reduced the survival
rate. As a rule, both TSA and BuA were more effective in the short-lived line
than in the long-lived line. It appears that different genetic background may
determine the action as well as the type and optimal concentrations of the
HDAC inhibitors. Furthermore, combined treatments of both inhibitors and heat
shock (TSA-RMHS and BuA-RMHS) and heat shock alone (RMHS) had basically the
same effect on life extension (Fig.
5D,H). We reason that the life extension by RMHS treatment may
mask the influences of HDAC inhibitors. These results suggest that
establishment of an altered cellular environment by HDAC inhibitors and heat
shock changed the lifespan of flies, possibly through a pathway that involves
the change in expression of certain genes resulting in inhibition of the
accumulation of damaged proteins, and/or stimulation of repair mechanisms.

ACKNOWLEDGEMENTS

This work was supported by grants from The National Natural Science
Foundation of China (30370316) and The Major State Basic Research Project of
China (G1999053902).

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